Modulation of immunostimulatory activity of immunostimulatory oligonucleotide analogs by positional chemical changes

ABSTRACT

The invention relates to the therapeutic use of oligonucleotides or oligonucleotide analogs as immunostimulatory agents in immunotherapy applications. The invention provides methods for enhancing the immune response caused by immunostimulatory oligonucleotide compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/965,116, filed on Sep. 26, 2001 (now U.S. Pat. No. 7,262,286), whichis a continuation-in-part of U.S. patent application Ser. No. 09/712,898(now abandoned), filed on Nov. 15, 2000, and which also claims priorityfrom the benefit of U.S. provisional patent application Ser. Nos.60/235,452 and 60/235,453, both filed on Sep. 26, 2000. Each of thepatent applications listed above is hereby incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the therapeutic use of oligonucleotides oroligonucleotide analogs as immunostimulatory agents in immunotherapyapplications.

2. Summary of the Related Art

Oligonucleotides have become indispensable tools in modern molecularbiology, being used in a wide variety of techniques, ranging fromdiagnostic probing methods to PCR to antisense inhibition of geneexpression and immunotherapy applications. This widespread use ofoligonucleotides has led to an increasing demand for rapid, inexpensiveand efficient methods for synthesizing oligonucleotides.

The synthesis of oligonucleotides for antisense and diagnosticapplications can now be routinely accomplished. See e.g., Methods inMolecular Biology, Vol 20: Protocols for Oligonucleotides and Analogspp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides andAnalogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991);and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech.6: 12 (1995); and Antisense Research and Applications (Crooke andLebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approachesincluded phosphodiester and phosphotriester chemistries. Khorana et al.,J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry foroligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179(1978), discloses phosphotriester chemistry for synthesis ofoligonucleotides and polynucleotides. These early approaches havelargely given way to the more efficient phosphoramidite andH-phosphonate approaches to synthesis. Beaucage and Caruthers,Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use ofdeoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawaland Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimizedsynthesis of oligonucleotides by the H-phosphonate approach.

Both of these modern approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al., Biochemistry 23: 3443 (1984), disclosessynthesis of oligonucleotide phosphorothioates using phosphoramiditechemistry. Jager et al., Biochemistry 27: 7237 (1988), disclosessynthesis of oligonucleotide phosphoramidates using phosphoramiditechemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083(1988), discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

More recently, several researchers have demonstrated the validity of theuse of oligonucleotides as immunostimulatory agents in immunotherapyapplications. The observation that phosphodiester and phosphorothioateoligonucleotides can induce immune stimulation has created interest indeveloping this side effect as a therapeutic tool. These efforts havefocused on phosphorothioate oligonucleotides containing the dinucleotideCpG.

Kuramoto et al., Jpn. J. Cancer Res. 83: 1128-1131 (1992) teaches thatphosphodiester oligonucleotides containing a palindrome that includes aCpG dinucleotide can induce interferon-alpha and gamma synthesis andenhance natural killer activity. Krieg et al., Nature 371: 546-549(1995) discloses that phosphorothioate CpG-containing oligonucleotidesare immunostimulatory. Liang et al., J. Clin. Invest. 98: 1119-1129(1996) discloses that such oligonucleotides activate human B cells.

Pisetsky, D. S.; Rich C. F., Life Sci. 54: 101 (1994), teaches that theimmunostimulatory activity of CpG-oligos is further enhanced by thepresence of phosphorothioate (PS) backbone on these oligos. Tokunaga,T.; Yamamoto, T.; Yamamoto, S. Jap. J. Infect. Dis. 52: 1 (1999),teaches that immunostimulatory activity of CpG-oligos is dependent onthe position of CpG-motif and the sequences flanking CpG-motif. Themechanism of activation of immune stimulation by CpG-oligos has not beenwell understood. Yamamoto, T.; Yamamoto, S.; Kataoka, T.; Tokunaga, T.,Microbiol. Immunol. 38: 831 (1994), however, suggests that CpG-oligostrigger immune cascade by binding to an intracellular receptor/protein,which is not characterized yet.

Several researchers have found that this ultimately triggers stresskinase pathways, activation of NF-κB and induction of various cytokinessuch as IL-6, IL-12, γ-IFN, and TNF-α. (See e.g., Klinman, D. M.; Yi, A.K.; Beaucage, S. L.; Conover, J.; Krieg, A. M., Proc. Natl. Acad. Sci.U.S.A. 93: 2879 (1996); Sparwasser, T.; Miethke, T.; Lipford, G. B.;Erdmann, A.; Haecker, H.; Heeg, K.; Wagner, H., Eur. J. Immunol. 27:1671 (1997); Lipford, G. B.; Sparwasser, T.; Bauer, M.; Zimmermann, S.;Koch, E. S.; Heeg, K.; Wagner, H. Eur. J., Immunol. 27: 3420 (1997);Sparwasser, T.; Koch, E. S.; Vabulas, R. M.; Lipford, G. B.; Heeg, K.;Ellart, J. W.; Wagner, H., Eur. J. Immunol. 28: 2045 (1998); and Zhao,Q.; Temsamani, J.; Zhou, R. Z.; Agrawal, S. Antisense Nucleic Acid DrugDev. 7: 495 (1997).)

The use of CpG-PS-oligos as antitumor, antiviral, antibacterial andantiinflammatory agents and as adjuvants in immunotherapy has beenreported. (See e.g., Dunford, P. J.; Mulqueen, M. J.; Agrawal, S.Antisense 97: Targeting the Molecular Basis of Disease, (NatureBiotechnology) Conference abstract, 1997, pp 40; Agrawal, S.; KandimallaE. R. Mol. Med. Today 6: 72 (2000); Chu. R. S.; Targoni, O. S.; Krieg,A. M.; Lehmann, P. V.; Harding, C. V. J. Exp. Med. 186: 1623 (1997);Zimmermann, S.; Egeter, O.; Hausmann, S.; Lipford, G. B.; Rocken, M.;Wagner, H.; Heeg, K. J. Immunol. 160: 3627 (1998).) Moldoveanu et al.,Vaccine 16: 1216-124 (1998) teaches that CpG-containing phosphorothioateoligonucleotides enhance immune response against influenza virus.McCluskie and Davis, J. Immunol. 161: 4463-4466 (1998) teaches thatCpG-containing oligonucleotides act as potent adjuvants, enhancingimmune response against hepatitis B surface antigen.

Zhao, Q.; Temsamani, J.; Idarola, P.; Jiang, Z.; Agrawal, S. Biochem.Pharmacol. 51: 173 (1996), teaches that replacement of deoxynucleosidesin a CpG-motif with 2′-O-methylribonucleosides suppressesimmunostimulatory activity, suggesting that a rigid C3′-endoconformation induced by 2′-O-methyl modification does not allow properrecognition and/or interaction of CpG-motif with the proteins involvedin the immunostimulatory pathway. This reference further teaches thatsubstitution of a methyl group for an unbridged oxygen on the phosphategroup between C and G of a CpG-motif suppresses immune stimulatoryactivity, suggesting that negative charge on phosphate group isessential for protein recognition and interaction.

Zhao, Q.; Yu, D.; Agrawal, S. Bioorg. Med. Chem. Lett. 9: 3453 (1999),teaches that substitution of one or two 2′-deoxynucleosides adjacent toCpG-motif with 2′- or 3′-O-methylribonucleosides on the 5′-side causes adecrease in immunostimulatory activity, while the same substitutionshave insignificant effect when they were placed on the 3′-side of theCpG-motif. However, Zhao, Q.; Yu, D.; Agrawal, S. Bioorg. Med. Chem.Lett. 10: 1051 (2000), teaches that the substitution of adeoxynucleoside two or three nucleosides away from the CpG-motif on the5′-side with one or two 2′-O-methoxyethyl- or 2′- or3′-O-methylribonucleosides results in a significant increase inimmunostimulatory activity.

The precise structural requirements and specific functional groups ofCpG-motif necessary for the recognition of protein/receptor factor thatis responsible for immune stimulation have not yet been studied indetail. There is, therefore, a need for new immunostimulatory motifswhich may provide improved immunostimulatory activity.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for enhancing the immune response causedby immunostimulatory oligonucleotide compounds. The methods according tothe invention enable increasing the immunostimulatory effect forimmunotherapy applications. Thus, the invention further provides methodsfor making and using such oligonucleotide compounds.

The present inventors have surprisingly discovered that positionalmodification of immunostimulatory oligonucleotides dramatically affectstheir immunostimulatory capabilities. In particular, modifications inthe immunostimulatory domain and/or the potentiation domain enhance theimmunostimulatory effect in a reproducible and predictable manner.

In a first aspect, the invention provides immunostimulatoryoligonucleotide compounds comprising an immunostimulatory domain and,optionally, one or more potentiation domains. In some embodiments, theimmunostimulatory domain comprises a dinucleotide analog that includes anon-naturally occurring pyrimidine base. In some embodiments, theimmunostimulatory domain and/or the potentiation domain include animmunostimulatory moiety at a specified position, as describedhereinbelow. In some embodiments, the immunostimulatory oligonucleotidecomprises a 3′-3′ linkage. In one embodiment, such 3′-3′ linkedoligonucleotides have two accessible 5′-ends.

In a second aspect, the invention provides methods for modulating theimmunostimulatory effect of an immunostimulatory oligonucleotidecompound. In some embodiments, the method comprises introducing into theimmunostimulatory domain a dinucleotide analog that includes anon-naturally occurring pyrimidine base. In some embodiments, the methodcomprises introducing into the immunostimulatory domain and/orpotentiation domain an immunostimulatory moiety at a specified position,as described hereinbelow. In some embodiments, the method comprisesintroducing into the oligonucleotide a 3′-3′ linkage.

In a third aspect, the invention provides methods for generating animmune response in a patient, such methods comprising administering tothe patient an immunostimulatory oligonucleotide compound according tothe invention.

In a fourth aspect, the invention provides methods for therapeuticallytreating a patient having disease caused by a pathogen, such methodscomprising administering to the patient an immunostimulatoryoligonucleotide compound according to the invention.

In a fifth aspect, the invention provides methods for treating a cancerpatient, such methods comprising administering to the patient animmunostimulatory oligonucleotide compound according to the invention.

In a sixth aspect, the invention provides methods for treatingautoimmune disorders, such as autoimmune asthma, such methods comprisingadministering to the patient an oligonucleotide analog immunostimulatorycompound according to the invention. Administration is carried out asdescribed for the third aspect of the invention.

In a seventh aspect, the invention provides methods for treating airwayinflammation or allergies, such methods comprising administering to thepatient an oligonucleotide analog immunostimulatory compound accordingto the invention. Administration is carried out as described for thethird aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B shows results of proliferation assays using oligonucleotides(SEQ ID NOs:8-23) having 1′,2′-dideoxyribose substitutions at variouspositions.

FIGS. 2A-2B shows results of spleen weight assays using oligonucleotides(SEQ ID NOs:8-23) having 1′,2′-dideoxyribose substitutions at variouspositions.

FIGS. 3A-3B shows results of proliferation assays using differenceoligonucleotides (SEQ ID NOs:1, 105-110) having 1′,2′-dideoxyribosesubstitutions at various positions.

FIGS. 4A-4B shows results of spleen weight assays using differentoligonucleotides (SEQ ID NOs:1, 105-110) having 1′,2′-dideoxyribosesubstitutions at various positions.

FIGS. 5A-5B shows results of proliferation assays using oligonucleotides(SEQ ID NOs:1, 8, 24-34) having C3-linker substitutions at variouspositions.

FIGS. 6A-6B shows results of spleen weight assays using oligonucleotides(SEQ ID NOs:1, 8, 24-34) having C3-linker substitutions at variouspositions.

FIGS. 7A-7B shows results of proliferation assays using oligonucleotides(SEQ ID NOs:1, 8, 35-42) having Spacer 9 or Spacer 18 substitutions atvarious positions.

FIGS. 8A-8B shows results of spleen weight assays using oligonucleotides(SEQ ID NOs:1, 8, 35-42) having Spacer 9 or Spacer 18 substitutions atvarious positions.

FIGS. 9A-9B shows results of proliferation assays using oligonucleotides(SEQ ID NOs:1, 43-47) having amino-linker substitutions at variouspositions.

FIGS. 10A-10B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 43-47) having amino-linker substitutionsat various positions.

FIGS. 11A-11B shows results of proliferation assays usingoligonucleotides (SEQ ID Nos:1, 8, 48-56) having 3′-deoxynucleosidesubstitutions at various positions.

FIGS. 12A-12B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 8, 48-56) having 3′-deoxynucleosidesubstitution at various positions.

FIGS. 13A-13B shows results of proliferation assays usingoligonucleotides (SEQ ID NOs:1, 57-68) having methylphosphonatesubstitutions at various positions.

FIGS. 14A-14B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 57-68) having methylphosphonatesubstitutions at various positions.

FIGS. 15A-15B shows results of proliferation assays usingoligonucleotides (SEQ ID NOs:69-72) having 2′-O-methylribonucleoside or2′-O-methoxyethyl substitutions at various positions.

FIGS. 16A-16B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:69-72) having 2′-O-methylribonucleoside or2′-O-methoxyethyl substitutions at various positions.

FIGS. 17A-17C shows results of proliferation assays usingoligonucleotides (SEQ ID NOs:73-80) having 5′-3′,5′-5′, or 3′-3′ linkagesubstitutions at various positions.

FIGS. 18A-18B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 81-88) having 13-L-deoxynucleotidesubstitutions at various positions.

FIGS. 19A-19B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 89-90) having 2′-O-propargylsubstitutions at various positions.

FIGS. 20A-20B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs: δ 91-95) having various substitution atvarious positions.

FIGS. 21A-21C shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 96-100) having 7-deazaguaninesubstitution within the immunostimulatory dinucleotide.

FIGS. 22A-22B shows results of proliferation assays usingoligonucleotides (SEQ ID NOs:1, 101, 102) having 6-thioguaninesubstitution within the immunostimulatory dinucleotide.

FIGS. 23A-23B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1-5) having 5-hydroxycytosine orN4-ethylcytosine substitution within the immunostimulatory dinucleotide.

FIGS. 24A-24B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1-5) having 5-hydroxycytosine orN4-ethylcytosine substitution within the immunostimulatory dinucleotide.

FIGS. 25A-25B shows results of proliferation assays usingoligonucleotides (SEQ ID NOs:1, 111-112) having arabinofuranosylcytosine(aracytidine: Ara-C) substitution within the immunostimulatorydinucleotide.

FIGS. 26A-26B shows results of spleen weight assays usingoligonucleotides (SEQ ID NOs:1, 103-104) having 4-thiouracilsubstitution within the immunostimulatory dinucleotide.

FIG. 27 shows the chemical structure of a CpG-motif, showing functionalgroups on cytosine that serve as hydrogen bond acceptor and hydrogenbond donor groups.

FIG. 28 shows the chemical structures of cytosine (1) and cytosineanalogs (2-7). In the nucleosides cytidine, deoxycytidine, and relatedanalogs, the substituent R is ribose or 2′-deoxyribose.

DETAILED DESCRIPTION

The invention relates to the therapeutic use of oligonucleotides andoligonucleotide analogs as immunostimulatory agents for immunotherapyapplications. The patents and publications cited herein reflect thelevel of knowledge in the field and are hereby incorporated by referencein their entirety. In the event of conflict between any teaching of anyreference cited herein and the present specification, the latter shallprevail, for purposes of the invention.

The invention provides methods for enhancing the immune response causedby immunostimulatory oligonucleotide compounds for immunotherapyapplications. Thus, the invention further provides compounds havingoptimal levels of immunostimulatory effect for immunotherapy and methodsfor making and using such oligonucleotide compounds.

The present inventors have surprisingly discovered that positionalchemical modifications introduced in immunostimulatory oligonucleotidesdramatically affect their immunostimulatory capabilities. In particular,modifications in the immunostimulatory domain and/or the potentiationdomain can enhance the immunostimulatory effect in a reproducible mannerfor desired applications.

In a first aspect, the invention provides immunostimulatoryoligonucleotide compounds comprising an immunostimulatory domain and,optionally, one or more potentiation domains. In certain preferredembodiments, the immunostimulatory domain comprises a dinucleotideanalog that includes a non-natural pyrimidine nucleoside.

For purposes of all aspects of the invention, the term “oligonucleotide”includes polymers of two or more deoxyribonucleosides, or any modifiednucleoside, including 2′- or 3′-substituted nucleosides, 2′- or3′-O-substituted ribonucleosides, deazanucleosides, or any combinationthereof. Such monomers may be coupled to each other by any of thenumerous known internucleoside linkages. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphotriester, phosphorothioate, phosphorodithioate, orphosphoramidate linkages, including 3′-5′, 2′-5′, 3′-3′, and 5′-5′linkages of any of the foregoing, or combinations thereof. The termoligonucleotide also encompasses such polymers having chemicallymodified bases or sugars and/or having additional substituents,including without limitation lipophilic groups, intercalating agents,diamines and adamantane. The term oligonucleotide also encompassespeptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholinonucleic acids, andoligonucleotides comprising non-pentose sugar (e.g. hexose) or abasicsugar backbones or backbone sections, as well as oligonucleotides thatinclude backbone sections with non-sugar linker or spacer groups, asfurther described hereinbelow.

For purposes of the invention the terms “2′-substituted” and“3′-substituted” mean (respectively) substitution of the 2′ (or 3′)position of the pentose moiety with a halogen (preferably Cl, Br, or F),or an —O-lower alkyl group containing 1-6 saturated or unsaturatedcarbon atoms, or with an —O-aryl or allyl group having 2-6 carbon atoms,wherein such alkyl, aryl or allyl group may be unsubstituted or may besubstituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro,acyl, acyloxy, alkoxy, carboxyl, carbalkoxy, or amino groups; or such 2′substitution may be with a hydroxy group (to produce a ribonucleoside)or an amino group, but not with a 2′ (or 3′) H group.

For purposes of the invention, the term “immunostimulatoryoligonucleotide compound” means a compound comprising animmunostimulatory dinucleotide, without which the compound would nothave an immunostimulatory effect. An “immunostimulatory dinucleotide” isa dinucleotide having the formula 5′-pyrimidine-purine-3′, wherein“pyrimidine” is a natural or non-natural pyrimidine nucleoside and“purine” is a natural or non-natural purine nucleoside. One suchimmunostimulatory dinucleotide is CpG. The terms “CpG” and “CpGdinucleotide” refer to the dinucleotide5′-deoxycytidine-deoxyguanosine-3′, wherein p is an internucleotidelinkage, preferably selected from the group consisting ofphosphodiester, phosphorothioate, and phosphorodithioate.

For purposes of the invention, a “dinucleotide analog” is animmunostimulatory dinucleotide as described above, wherein either orboth of the pyrimidine and purine nucleosides is a non-naturalnucleoside. A “non-natural” nucleoside is one that includes anon-naturally occurring base and/or a non-naturally occurring sugarmoiety. For purposes of the invention, a base is considered to benon-natural if it is not selected from the group consisting of thymine,guanine, cytosine, adenine, and uracil. The terms “C*pG” and “CpG*”refer to immunostimulatory dinucleotide analogs comprising a cytidineanalog (non-natural pyrimidine nucleoside) or a guanosine analog(non-natural purine nucleoside), respectively.

FIG. 27 shows the chemical structure of a CpG-motif, showing thefunctional groups on cytosine that serve as hydrogen bond acceptor andhydrogen bond donor groups. Cytosine has two hydrogen bond acceptorgroups at positions 2 (keto-oxygen) and 3 (nitrogen), and a hydrogenbond donor group at the 4-position (amino group) These groups can serveas potential recognizing and interacting groups with receptors that areresponsible for immune stimulation. FIG. 28 shows cytosine analogs thatare isostructural with natural cytosine, including5-methyl-deoxycytosine (2), 5-methyl-deoxyisocytosine (3),5-hydroxy-deoxycytosine (4), deoxyuridine (5), N4-ethyl-deoxycytosine(6), and deoxy-P-base (7).

In one embodiment, therefore, the immunostimulatory dinucleotidecomprises a pyrimidine nucleoside of structure (I):

wherein D is a hydrogen bond donor, D′ is selected from the groupconsisting of hydrogen, hydrogen bond donor, hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group, A is a hydrogen bond acceptor, X is carbon ornitrogen, and S is a pentose or hexose sugar ring linked to thepyrimidine base. In some embodiments, the pyrimidine nucleoside is anon-natural pyrimidine nucleoside, i.e., the compound of structure (I)is not cytidine or deoxycytidine.

In some embodiments, the base moiety in (I) is a non-naturally occurringpyrimidine base. Examples of preferred non-naturally occurringpyrimidine bases include, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine,and 4-thiouracil. In some embodiments, the sugar moiety S in (I) is anon-naturally occurring sugar moiety. For purposes of the presentinvention, a “naturally occurring sugar moiety” is ribose or2′-deoxyribose, and a “non-naturally occurring sugar moiety” is anysugar other than ribose or 2′-deoxyribose that can be used in thebackbone for an oligonucleotide. Arabinose and arabinose derivatives areexamples of a preferred non-naturally occurring sugar moieties.

Immunostimulatory domains according to the invention may includeimmunostimulatory moieties on one or both sides of the immunostimulatorynatural dinucleotide or non-natural dinucleotide analog. For example, animmunostimulatory domain could be depicted as

5′ - - - X1-X2-Y-Z-X3-X4 - - - 3′wherein Y represents cytidine or a non-natural pyrimidine nucleosideanalog, Z represents guanosine or a non-natural purine nucleosideanalog, and each X independently represents a nucleoside or animmunostimulatory moiety according to the invention. An“immunostimulatory moiety” is a chemical structure at a particularposition within the immunostimulatory domain or the potentiation domainthat causes the immunostimulatory oligonucleotide to be moreimmunostimulatory than it would be in the absence of theimmunostimulatory moiety.

Preferred immunostimulatory moieties include modifications in thephosphate backbones including without limitation methylphosphonates,methylphosphonothioates phosphotriesters, phosphothiotriestersphosphorothioates, phosphorodithioates, triester prodrugs, sulfones,sulfonamides, sulfamates, formacetal, N-methylhydroxylamine, carbonate,carbamate, boranophosphonate, phosphoramidates, especially primaryamino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, andstereospecific linkages (e.g., (R)- or (S)-phosphorothioate,alkylphosphonate, or phosphotriester linkages). Preferredimmunostimulatory moieties according to the invention further includenucleosides having sugar modifications, including without limitation2′-substituted pentose sugars including without limitation2′-O-methylribose, 2′-O-methoxyethylribose, 2′-O-propargylribose, and2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, includingwithout limitation 3′-O-methylribose; 1′,2′-dideoxyribose; hexosesugars, including without limitation arabinose, 1′-methylarabinose,3′-hydroxymethylarabinose, 4′-hydroxymethylarabinose, and 2′-substitutedarabinose sugars; and alpha-anomers.

Preferred immunostimulatory moieties according to the invention furtherinclude oligonucleotides having other carbohydrate backbonemodifications and replacements, including peptide nucleic acids (PNA),peptide nucleic acids with phosphate groups (PHONA), locked nucleicacids (LNA), morpholinonucleic acids, and oligonucleotides havingbackbone sections with alkyl linkers or amino linkers. The alkyl linkermay be branched or unbranched, substituted or unsubstituted, andchirally pure or a racemic mixture. Most preferably, such alkyl linkershave from about 2 to about 18 carbon atoms. In some preferredembodiments such alkyl linkers have from about 3 to about 9 carbonatoms. Such alkyl linkers include polyethyleneglycol linkers[—O—CH2-CH2-]_(n) (n=2-9). In some preferred embodiments, such alkyllinkers may include peptides or amino acids.

Preferred immunostimulatory moieties according to the invention furtherinclude DNA isoforms, including without limitation β-L-deoxynucleosidesand alpha-deoxynucleosides. Preferred immunostimulatory moietiesaccording to the invention further include nucleosides having unnaturalinternucleoside linkage positions, including without limitation2′-5′,2′-2′,3′-3′ and 5′-5′ linkages.

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having modified heterocyclic bases, includingwithout limitation 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine,N4-alkyldeoxycytidine, preferably N4-ethyldeoxycytidine, 4-thiouridine,6-thiodeoxyguanosine, 7-deazaguanosine, and deoxyribonucleosides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine, includingwithout limitation 2,6-diaminopurine.

By way of specific illustration and not by way of limitation, forexample, in the immunostimulatory domain described earlier

5′ - - - X1-X2-Y-Z-X3-X4 - - - 3′a nucleoside methylphosphonate at position X3 or X4 is animmunostimulatory moiety, a substituted or unsubstituted alkyl linker atposition X1 is an immunostimulatory moiety, and a β-L-deoxynucleoside atposition X1 is an immunostimulatory moiety. See Table 1 below forrepresentative positions and structures of immunostimulatory moietieswithin the immunostimulatory domain.

TABLE 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES X1 C3-alkyl linker,2-aminobutyl-1,3-propanediol linker (amino linker), β-L-deoxynucleosideX2 2-aminobutyl-1,3-propanediol linker X3 nucleoside methylphosphonateX4 nucleoside methylphosphonate, 2′-O-methyl-ribonucleoside

In some embodiments, the immunostimulatory oligonucleotide furthercomprises a potentiation domain

A “potentiation domain” is a region of an immunostimulatoryoligonucleotide analog, other than the immunostimulatory domain, thatcauses the oligonucleotide to be more immunostimulatory if it containsthe potentiation domain than the oligonucleotide would be in the absenceof the potentiation domain. The potentiation domain can be upstream ordownstream relative to the immunostimulatory domain. The term “upstream”is used to refer to positions on the 5′ side of the immunostimulatorydinucleotide or dinucleotide analog (Y-Z). The term “downstream” is usedto refer to positions on the 3′ side of Y-Z.

For example, an immunostimulatory oligonucleotide analog could have thestructure

5′-U9-U8-U7-U6-U5-U4-U3-U2-U1-X1-X2-Y-Z-X3-X4-N-N-N-3′wherein U9-U1 represents an upstream potentiation domain, wherein each Uindependently represents the same or a different nucleosideimmunostimulatory moiety, N represents any nucleoside and X1-X4, Y and Zare as before.

Alternatively, an immunostimulatory oligonucleotide analog could havethe structure

5′-N-N-X1-X2-Y-Z-X3-X4-D1-D2-D3-D4-D5-D6-D7-D8-3′wherein D1-D8 represents a downstream potentiation domain, wherein eachD independently represents the same or a different nucleoside orimmunostimulatory moiety, and all other symbols are as described above.

In these configurations, an immunostimulatory moiety at U6 would be sixpositions upstream from the immunostimulatory dinucleotide ordinucleotide analog and an immunostimulatory moiety at D4 would be fourpositions downstream from the immunostimulatory dinucleotide ordinucleotide analog. The term “position” is used rather than“nucleoside”, because any of the U or D positions can represent animmunostimulatory moiety which may or may not be a nucleoside ornucleoside analog. Of course, oligonucleotide analogs can be constructedhaving both upstream and downstream potentiation domains.

Table 2 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving an upstream potentiation domain. See FIG. 7 for definitions ofSpacer 9 and Spacer 18 as referred to in Tables 2 and 3.

TABLE 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY X22-aminobutyl-1,3-propanediol linker X1 C3-linker,2-aminobutyl-1,3-propanediol linker, β-L- deoxynucleoside U11′,2′-dideoxyribose, C3-linker, 2′-O-methyl-ribonucleoside U21′,2′-dideoxyribose, C3-linker, Spacer 18, 3′-deoxynucleoside,nucleoside methylphosphonate, β-L-deoxynucleoside, 2′-O-propargyl-ribonucleoside U3 1′,2′-dideoxyribose, C3-linker, Spacer 9,Spacer 18, nucleoside methylphosphonate, 2′-5′ linkage U2 + U31′,2′-dideoxyribose, C3-linker,, β-L-deoxynucleoside U3 + U4 nucleosidemethylphosphonate, 2′-O-methoxyethyl- ribonucleoside U5 + U61′,2′-dideoxyribose, C3-linker X1 + U3 1′,2′-dideoxyribose

Table 3 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving a downstream potentiation domain.

TABLE 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY X3 nucleosidemethylphosphonate X4 nucleoside methylphosphonate,2′-O-methyl-ribonucleoside D1 1′,2′-dideoxyribose, nucleosidemethylphosphonate D2 1′,2′-dideoxyribose, C3-linker, Spacer 9, Spacer18, 2-aminobutyl-1,3-propanediol-linker, nucleoside methylphosphonate,β-L-deoxynucleoside D3 3′-deoxynucleoside,2′-O-propargyl-ribonucleoside, 2′-5′-linkage D2 + D31′,2′-dideoxyribose, β-L-deoxynucleoside

In another embodiment of the invention, the oligonucleotide according tothe invention has one or two accessible 5′ ends. The present inventorshave discovered that immunostimulatory moieties in the region 5′ to theimmunostimulatory dinucleotide have a greater impact onimmunostimulatory activity than do similar substitutions in the region3′ to the immunostimulatory dinucleotide. This observation suggests thatthe 5′-flanking region of CpG-PS-oligos plays an important role inimmunostimulatory activity. Moreover, the inventors have discovered thatcompounds having two oligonucleotide units attached by way of a 3′-5′ or3′-3′ linkage have greater immunostimulatory activity than do compoundsin which the two oligonucleotide units are attached by way of a 5′-5′linkage. In some preferred embodiments, therefore, the immunostimulatoryoligonucleotide according to the invention comprises a 3′-3′ linkage. Insome such embodiments, the oligonucleotides have one or two accessible5′ ends.

In a second aspect, the invention provides methods for modulating theimmunostimulatory effect of an immunostimulatory oligonucleotide. Insome embodiments, the method comprises introducing into theimmunostimulatory domain a dinucleotide analog that includes anon-naturally occurring pyrimidine base, as described above for thefirst aspect of the invention. In some embodiments, the method comprisesintroducing into the immunostimulatory domain and/or potentiation domainan immunostimulatory moiety at a specified position, as described above.In some embodiments, the method comprises introducing into theoligonucleotide a 3′-3′ linkage.

For purposes of the invention, “introducing an immunostimulatory moiety”at a specified position simply means synthesizing an oligonucleotidethat has an immunostimulatory moiety at the specified position. Forexample, “introducing an immunostimulatory moiety into position U6”simply means synthesizing an oligonucleotide that has animmunostimulatory moiety at such a position, with reference to, e.g.,the following structure:

5′-U9-U8-U7-U6-U5-U4-U3-U2-U1-X1-X2-Y-Z-X3-X4-D1-D2-D3-3′.

Preferably, the methods according to this aspect of the inventioninclude introducing an immunostimulatory moiety at a position in theimmunostimulatory domain or in an upstream or downstream potentiationdomain according to the preferred substitution patterns described inTables 1-3.

The methods according to this aspect of the invention can beconveniently carried out using any of the well-known synthesistechniques by simply using an appropriate immunomodulatory moietymonomer synthon in the synthesis process in an appropriate cycle toobtain the desired position. Preferred monomers includephosphoramidites, phosphotriesters and H-phosphonates. PS-oligos arereadily synthesized, e.g., using β-cyanoethylphosphoramidite chemistryon CPG solid support using appropriate phosphoramidites, deprotected asrequired, purified by C₁₈ reverse phase HPLC, dialyzed against distilledwater and lyophilized. The purity of each PS-oligo is readily determinedby CGE and the molecular weight can be confirmed by MALDI-TOF massspectral analysis.

In a third aspect, the invention provides methods for generating animmune response in a patient, such methods comprising administering tothe patient an oligonucleotide analog immunostimulatory compoundaccording to the invention.

In the methods according to this aspect of the invention, preferably,administration of compounds is parenteral, oral, sublingual,transdermal, topical, intranasal, intratracheal, intravaginal, orintrarectal. Administration of the therapeutic compositions can becarried out using known procedures at dosages and for periods of timeeffective to reduce symptoms or surrogate markers of the disease. Whenadministered systemically, the therapeutic composition is preferablyadministered at a sufficient dosage to attain a blood level ofoligonucleotide from about 0.001 micromolar to about 10 micromolar. Forlocalized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. Preferably,a total dosage of oligonucleotide will range from about 0.1 mgoligonucleotide per patient per day to about 40 mg oligonucleotide perkg body weight per day. It may be desirable to administersimultaneously, or sequentially a therapeutically effective amount ofone or more of the therapeutic compositions of the invention to anindividual as a single treatment episode. In some instances, dosagesbelow the above-defined ranges may still provide efficacy. In apreferred embodiment, after the composition of matter is administered,one or more measurement is taken of biological effects selected from thegroup consisting of complement activation, mitogenesis and inhibition ofthrombin clot formation.

In certain preferred embodiments, compounds according to the inventionare administered in combination with antibiotics, antigens, allergens,vaccines, antibodies, cytotoxic agents, antisense oligonucleotides, genetherapy vectors, DNA vaccines and/or adjuvants to enhance thespecificity or magnitude of the immune response. Either the compound orthe vaccine, or both may optionally be linked to an immunogenic protein,such as keyhole limpet hemocyanin, cholera toxin B subunit, or any otherimmunogenic carrier protein. Any of a plethora of adjuvants may be used,including, without limitation, Freund's complete adjuvant,monophosphoryl lipid A (MPL), saponins, including QS-21, alum, andcombinations thereof. Certain preferred embodiments of the methodsaccording to the invention induce cytokines by administration ofimmunostimulatory oligonucleotide compounds. In certain embodiments theimmunostimulatory oligonucleotide compounds are conjugated to anantigen, hapten, or vaccine. As discussed above, the present inventorshave discovered that an accessible 5′ end is important to the activityof certain immunostimulatory oligonucleotide compounds. Accordingly, foroptimum immunostimulatory activity, the oligonucleotide preferably isconjugated to an antigen or vaccine by means of the 3′-end ofoligonucleotide compound.

For purposes of this aspect “in combination with” means in the course oftreating the same disease in the same patient, and includesadministering the oligonucleotide and/or the vaccine and/or the adjuvantin any order, including simultaneous administration, as well astemporally spaced order of up to several days apart. Such combinationtreatment may also include more than a single administration of theoligonucleotide, and/or independently the vaccine, and/or independentlythe adjuvant. The administration of the oligonucleotide and/or vaccineand/or adjuvant may be by the same or different routes.

The method according to this aspect of the invention is useful for modelstudies of the immune system, and is further useful for the therapeutictreatment of human or animal disease.

In a fourth aspect, the invention provides methods for therapeuticallytreating a patient having disease caused by a pathogen, such methodscomprising administering to the patient an oligonucleotide analogimmunostimulatory compound according to the invention. Administration iscarried out as described for the third aspect of the invention.

In a fifth aspect, the invention provides methods for treating a cancerpatient, such methods comprising administering to the patient anoligonucleotide analog immunostimulatory compound according to theinvention. Administration is carried out as described for the thirdaspect of the invention.

In a sixth aspect, the invention provides methods for treatingautoimmune disorders, such as autoimmune asthma, such methods comprisingadministering to the patient an oligonucleotide analog immunostimulatorycompound according to the invention. Administration is carried out asdescribed for the third aspect of the invention.

In a seventh aspect, the invention provides methods for treating airwayinflammation or allergies, such methods comprising administering to thepatient an oligonucleotide analog immunostimulatory compound accordingto the invention. Administration is carried out as described for thethird aspect of the invention.

The following examples are intended to further illustrate certainpreferred embodiments of the invention, and are not intended to limitthe scope of the invention.

EXAMPLES Example 1 Synthesis of Oligonucleotides ContainingImmunomodulatory Moieties

Oligonucleotides were synthesized on a 1 micromolar scale using anautomated DNA synthesizer (Expedite 8909, PerSeptive Biosystems, FosterCity, Calif.). Standard deoxynucleoside phosphoramidites are obtainedfrom PerSeptive Biosystems. 1′,2′-dideoxyribose phosphoramidite,propyl-1-phosphoramidite, 2′-deoxy-5-nitroindole-ribofuranosylphosphoramidite, 2′-deoxy-uridine phosphoramidite, 2′-deoxy-Pphosphoramidite, 2′-deoxy-2-aminopurine phosphoramidite,2′-deoxy-nebularine phosphoramidite, 2′-deoxy-7-deazaguanosinephosphoramidite, 2′-deoxy-4-thiouridine phosphoramidite,2′-deoxy-isoguanosine phosphoramidite, 2′-deoxy-5-methylisocytosinephosphoramidite, 2′-deoxy-4-thiothymidine phosphoramidite,2′-deoxy-K-phosphoramidite, 2′-deoxy-2-aminoadenosine phosphoramidite,2′-deoxy-N4-ethyl-cytosine phosphoramidite, 2′-deoxy-6-thioguanosinephosphoramidite, 2′-deoxy-7-deaza-xanthosine phosphoramidite,2′-deoxy-8-bromoguanosine phosphoramidite, 2′-deoxy-8-oxoguanosinephosphoramidite, 2′-deoxy-5-hydroxycytosine phosphoramidite,arabino-cytosine phosphoramidite and 2′-deoxy-5-propynecytosinephosphoramidite were obtained from Glen Research (Sterling, Va.).2′-Deoxy-inosine phosphoramidite were obtained from ChemGenes (Ashland,Mass.).

Normal coupling cycles or a coupling cycle recommended by thephosphoramidite manufacturer were used for all phosphoramidites.Beaucage reagent was used as an oxidant to obtain phosphorothioatemodification. After synthesis, oligonucleotides were deprotected byincubating CPG-bound oligonucleotide with concentrated ammoniumhydroxide solution for 1.5-2 hours at room temperature and thenincubating the ammonium hydroxide supernatant for 12 hours at 55 degreesC. or as recommended by phosphoramidite manufacturer. The ammoniumhydroxide solution was evaporated to dryness in a speed-vac and5′-DMTr-oligonucleotides were purified by HPLC on a C18 reverse-phasematrix using a solvent system of 0.1 M ammonium acetate and 1:5 ratio0.1 M ammonium acetate in acetonitrile. Then the oligonucleotides weretreated with 80% acetic acid to remove the DMTr group, converted tosodium form and desalted by dialysis against double distilled water.Oligonucleotides were filtered through 0.4μ filters, lyophilized andredissolved in double distilled water. Characterization was achieved bydenaturing PAGE and MALDI-TOF mass spectrometry.

Example 2 Synthesis of CpG-PS-Oligos Containing Cytosine Analogs

Following the procedures outlined in Example 1, the followingoligonucleotides were synthesized:

Oligo # (SEQ ID NO): Sequence (5′--->3′) and Modification^(a) 1d(CTATCTGACGTTCTCTGT) 2 d(CTATCTGAC*GTTTCTCTGT) 3 d(CTATCTGACC*TTCTCTGT)4 d(CTATCTGAC*GTTCTCTGT) 5 d(CTATCTGACC*TTCTCTGT) ^(a)CpG-motif is shownin bold. C* represents 5-hydroxycytosine (oligos 2 and 3) orN4-ethylcytosine (oligos 4 and 5).

The oligonucleotides were characterized by CGE and MALDI-TOF massspectrometry (Brucker Proflex III MALDI-TOF mass spectrometer with 337nm N2 laser). Molecular weights observed and calculated (shown inparentheses) for each oligonucleotide are as follows: Oligo 1, 5704(5704.8); Oligo 2, 5720 (5720.8); Oligo 3, 5681 (5680.7); Oligo 4, 5733(5733); Oligo 5, 5694 (5693).

Example 3 Analysis of Spleen Weights in Treated Mice

Female BALB/c mice (4-5 weeks, 19-21 g, Charles River, Wilmington,Mass.) were used in the study. The animals were fed with commercial dietand water ad lib. The animals were injected intraperitoneally with 5 or10 mg/kg dose of immunostimulatory oligonucleotide compound dissolved insterile PBS. One group of mice received PBS alone to serve as a control(PBS). Four animals were used for each immunostimulatory oligonucleotidecompound. Mice were sacrificed 72 h later, spleens were harvested andweighed.

Example 4 Analysis of Immunostimulatory Oligonucleotide Compounds inMouse Lymphocyte Proliferation Assay

Spleens from CD-1, BALB/c, C57BL/6 mouse (4-8 weeks) were used as sourceof lymphocytes. Single cell suspensions were prepared by gently mincingwith the frosted ends of glass slides. Cells were then cultured in RPMIcomplete medium [RPMI medium supplemented with 10% fetal bovine serum(FBS) (heat-inactivated at 56° C. for 30 min), 50 μM 2-mercaptoethanol,100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine]. Thecells were then plated in 96-well dishes at a density of 10⁶ cells/mL ina final volume of 100 μL. Immunostimulatory oligonucleotide compounds orLPS (lipopolysaccharide) were added to the cell culture in 10 μL of TEbuffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). The cells were then set toculture at 37° C. After 44 h, 1 μCi ³H-uridine (Amersham, ArlingtonHeights, Ill.) was added to the culture in 20 μL of RPMI medium, and thecells were pulse-labeled for another 4 h. The cells were harvested byautomatic cell harvester (Skatron, Sterling, Va.), and the filters werecounted by a scintillation counter. The experiments were performed intriplicate.

Example 5 Lymphocyte Proliferatory Activity of CpG-PS-Oligos ContainingCytosine Analogs

The immunostimulatory activity of CpG-PS-oligos 1-5 (Example 4) wasstudied using a BALB/c mouse lymphocyte proliferation assay. In brief,mouse spleen cells were cultured and incubated with CpG-PS-oligos at0.1, 0.3, 1.0 and 3.0 μg/mL concentration for 48 hr and cellproliferation was measured by ³H-uridine incorporation.

FIG. 23 shows the dose-dependent cell proliferatory activity of oligos1-5 in mouse lymphocyte cultures. At a dose of 3.0 μg/mL, oligo 1, withnatural cytidine, showed a proliferation index of 29.5±2.1. Oligo 2, inwhich the cytosine base of the deoxycytidine of the CpG-motif isreplaced with a 5-hydroxycytosine, also showed dose-dependent lymphocyteproliferation. A proliferation index of 23.7±2.9 at 3.0 μg/mL dose wasobserved for oligo 2. PS-Oligo 4, which contained N4-ethyl-cytosine inplace of the cytosine base in the CpG-motif, also showed dose-dependentcell-proliferation activity. The proliferation index of 18.7±1.6observed for oligo 4 at a dose of 3 μg/mL suggests that the presence ofa bulky hydrophobic substitution on the 4-amino group of cytosine in aCpG-motif slightly impedes immunostimulatory activity.

Oligo 3, in which 5-hydroxy-deoxycytidine was placed in thedeoxyguanosine position instead of the deoxycytidine position of theCpG-motif, showed a proliferation index that was similar to thatobserved for media control (FIG. 23). Similarly, the control Oligo 5 inwhich deoxyguanosine in the CpG-motif was substituted withN4-ethyldeoxycytidine, showed cell proliferation similar to that ofmedia control.

Other oligos, in which cytosine base in the CpG-motif was replaced with5-methyl-deoxycytosine (2; see FIG. 28), 5-methyl-deoxyisocytosine (3),deoxyuridine (5), or deoxy-P-base (7) showed no or insignificant cellproliferatory activity in the same assay system. These results suggestthat (i) cell proliferatory activity is maintained when the cytosinebase of the CpG motif is replaced with 5-hydroxycytosine orN4-ethylcytosine (Oligos 2 and 4, respectively), but (ii) substitutionof the guanine base with these cytosine analogs results in a loss ofcell proliferatory activity.

Example 6 Splenomegaly in Mice Induced by CpG-PS-Oligos ContainingCytosine Analogs

To confirm the in vitro effects of CpG-PS-oligos, Oligos 1, 2, and 4(from Example 4) were injected intraperitoneally (ip) to BALB/c mice ata dose of 10 mg/kg and the change in spleen weight was measured as anindicator of the level of immunostimulatory activity of each PS-oligo.The change in spleen weight as a result of treatment with CpG-PS-oligosis presented in FIG. 24. Female BALB/c mice (4-6 weeks, 19-21 gm) weredivided in to different groups with four mice in each group.Oligonucleotides were dissolved in sterile PBS and administeredintraperitoneally to mice at a dose of 10 mg/kg. After 72 hr, mice weresacrificed and spleens were harvested and weighed. Each circlerepresents the spleen weight of an individual mouse and the + representsthe mean spleen weight for each group.

Oligo 1, which has natural deoxycytidine in the CpG-motif, showed about45% increase in spleen weight at a dose of 10 mg/kg, compared with thecontrol group of mice that received PBS. Oligo 2, which has a5-hydroxycytosine in place of the cytosine base in the CpG-motif, showedabout 35% increase in spleen weight at the same dose. Oligo 4, which hasN4-ethylcytosine in place of the cytosine base in the CpG-motif, showedabout 34% increase in spleen weight at the same dose compared to thecontrol group. These data confirm the results observed in lymphocyteproliferation assays for these oligos containing modified cytidineanalogs in place of deoxycytidine in the CpG-motif.

Example 7 Structure-Activity Relationships of C*pG-PS-Oligos

The presence of a methyl group at the 5-position of cytosine(5-methyl-deoxycytosine, 2 (FIG. 28)) in a CpG-motif completelyabolishes CpG related immunostimulatory effects of CpG-PS-oligos. Basedon the results observed in in vitro and in vivo experiments we haveconstructed structure-activity relationships for the PS-oligoscontaining cytosine analogs.

The replacement of the cytosine base (1) in the CpG-motif with5-methyl-isocytosine (3) resulted in complete loss of immunostimulatoryactivity, as is the case with 5-methylcytosine (2), which could be as aresult of switching the keto and amino groups at the 2 and 4-positions,respectively, and/or placing a hydrophobic methyl group at the5-position of cytosine.

Oligo 2, containing a hydrophilic hydroxy substitution at the 5-positionof the cytosine in the CpG-motif, showed immunostimulatory activitysimilar to that of oligo 1, which contains the natural cytosine base.This observation suggests that bulky hydrophilic groups are bettertolerated than are hydrophobic groups at the 5-position of cytosine forimmunostimulatory activity of CpG-PS-oligos. Perhaps the binding pocketfor the CpG-oligos on receptor is hydrophilic in nature and can notaccommodate a hydrophobic group at the 5-position of cytosine.

When the cytosine base in the CpG-motif is replaced with uracil (5 (seeFIG. 28)), in which keto groups are present at both the 2 and4-positions, no immunostimulatory activity was observed, suggesting thata hydrogen bond donor amino group at the 4-position of cytosine iscritical for immunostimulatory activity. When a large hydrophobic ethylgroup is placed on 4-amino group of cytosine in a CpG-motif, reducedlymphocyte proliferation and a slightly reduced increase in spleenweight in mice were observed, suggesting that a bulky ethyl group atthis position does not interfere with binding of the CpG-PS-oligo to thereceptor factors responsible for immunostimulatory activity. In spite ofthe ethyl substitution, the 4-amino group of N4-ethylcytosine (6) canparticipate in hydrogen bond formation with an acceptor. The modifiedpyrimidine base dP, in which the nitrogen group located at the4-position involved in ring structure formation with the 5-position, andwhich does not have a hydrogen bond donor amino group at the 4-position,had no mouse lymphocyte proliferation activity in cultures, suggestingthat the 4-amino group of cytosine in a CpG-motif is critical forimmunostimulatory activity.

In conclusion, the results presented here show that the functionalgroups at 2, 3, and 4 positions of the cytosine are important forCpG-related immunostimulatory activity. A hydrophobic substitution atthe 5-position of cytosine completely suppresses immunostimulatoryactivity of a CpG-oligo, while a hydrophilic group at this position istolerated well. In addition, the immunostimulatory activity ofCpG-PS-oligos containing 5-hydroxycytosine or N4-ethylcytosine in placeof cytosine in the CpG-motif can be modulated significantly byincorporating appropriate chemical modifications in the 5′-flankingsequence, suggesting that these cytosine analogs in a CpG-motif arerecognized as part of an immunostimulatory motif.

Example 8 Synthesis of End-Blocked CpG-PS Oligonucleotides

The CpG-PS-oligos shown in FIG. 17 were synthesized using an automatedsynthesizer and phosphoramidite approach. Oligo 1 (16-mer) wassynthesized using nucleoside-5′-β-cyanoethylphosphoramidites. Oligo 2, a32-mer, was synthesized using nucleoside-3′-β-cyanoethylphosphoramiditesand controlled pore glass support (CPG-solid support) with a 3′-linkednucleoside in which 16-mer sequence of Oligo 1 was repeated twice;therefore, Oligo 2 had two 16-mers (Oligo 1) linked by a normal3′-5′-linkage. Oligo 3, a 32-mer, was synthesized with two 16-mers(Oligo 1) linked by a 5′-5′-linkage, so Oligo 3 had two 3′-ends and no5′-end. Synthesis of Oligo 3 was carried out in two steps: the first16-mer was synthesized using nucleoside-3′-β-cyanoethylphosphoramiditesand solid support with a 3′-linked nucleoside, and then synthesis of thesecond 16-mer segment was continued usingnucleoside-5′-β-cyanoethylphosphoramidites. Oligo 4, a 32-mer, comprisedtwo 16-mers (Oligo 1) linked by a 3′-3′-linkage, so Oligo 4 had two5′-ends and no 3′-end. Synthesis of Oligo 4 was carried out in twosteps: the first 16-mer was synthesized usingnucleoside-5′-β-cyanoethylphosphoramidites and solid support with a5′-linked nucleoside, and the synthesis of the second 16-mer segment wascontinued using nucleoside-3′-β-cyanoethyl-phosphoramidites. Synthesisof Oligos 5-8 was carried out by using the samenucleoside-β-cyanoethylphosphoramidites as for Oligos 1-4, respectively.At the end of the synthesis, Oligos 1-8 were deprotected withconcentrated ammonia solution, purified by reversed phase HPLC,detritylated, desalted and dialyzed. The purity of each PS-oligo waschecked by CGE and the molecular weight was confirmed by MALDI-TOF massspectral analysis (Table 1). The sequence integrity and directionalityof 5′-CpG motif in Oligos 1-8 were confirmed by recording meltingtemperatures (T_(m)s) of the duplexes with their respective DNAcomplementary strands (5′-AAGGTCGAGCGTTCTC-3′ (SEQ ID NO: 6) for Oligos1-4, and 5′-ATGGCGCACGCTGGGAGA-3′ (SEQ ID NO: 7) for Oligos 5-8). TheT_(m)s of these duplexes were 53.9±0.9° C. (Oligos 1-4), 61.8° C. (Oligo5), and 58.8±0.6° C. (Oligos 6-8) (note that Oligo 5 was a 18-mer andOligos 6-8 were 32-mers but not 36-mers).

Example 9 Mouse Spleen Lymphocyte Proliferatory Activity of End-BlockedCpG-PS Oligonucleotides

Immunostimulatory activity of the end-blocked CpG-PS-oligos of Example 8was studied initially in a lymphocyte proliferation assay. Typically,mouse (Balb-C) spleen lymphocytes were cultured with CpG-PS-oligos atconcentrations of 0.1, 1.0, and 10.0 μg/ml for 48 h and cellproliferation was determined by ³H-uridine incorporation, as describedin Example 3. Results are shown in FIG. 17

Oligo 1 induced a dose-dependent effect on cell proliferation; at aconcentration of 10 μg/ml (˜2.0 μM), the proliferation index was5.0±0.32. Oligo 2, which consisted of two units of Oligo 1 linked by a3′-5′-linkage, had a proliferation index of 5.8±0.28 at the same dose(˜1.0 μM). Oligo 3, which consisted of two units of Oligo 1 linked by a5′-5′-linkage, had a proliferation index of 2.0±0.26, reflecting asignificantly lower immunostimulatory activity than observed with Oligos1 and 2. Oligo 4, which consisted of two units of Oligo 1 linked by a3′-3′-linkage, had a proliferation index of 7.2±0.5, reflecting agreater immunostimulatory activity than observed with Oligos 1 and 2.

Similar results were obtained with Oligos 5-8. Oligo 5 had aproliferation index of 3.9±0.12. Oligos 6-8, in which two units of Oligo5 are linked by a 3′-5′-linkage (Oligo 6), 5′-5′-linkage (Oligo 7), and3′-3′-linkage (Oligo 8) had proliferation indices of 4.9±0.2, 1.74±0.21,and 7.7±0.82, respectively. Comparison of the results obtained withOligos 6-8 show that Oligos 6 and 8, in which two Oligo 5 sequences werelinked by a 3′-5′-linkage or a 3′-3′-linkage had greaterimmunostimulatory activity, while Oligo 7, in which two Oligo 5 werelinked by a 5′-5′-linkage had significant less immunostimulatoryactivity, than did Oligo 5.

Based on lymphocyte proliferation results of Oligos 1-8, it is clearthat when oligos are linked through their 5′-ends, there is asignificant loss of immunostimulatory activity, while if they are linkedthrough their 3′-ends, there is an increase in immunostimulatoryactivity. It is important to note that 3′-3′-linked oligos have shownsubstantially greater stability towards degradation by exonucleases thanthe oligos that contained a free 3′-end, which could also result inincreased immunostimulatory activity. The lower immunostimulatoryactivity of Oligos 3 and 7, in which the 5′-end of oligos is blocked,suggests that accessibility to 5′-end of oligo is essential forimmunostimulatory activity of CpG-PS-oligos.

Example 10 Splenomegaly in Mice Induced by End-Blocked CpG-PSOligonucleotides

To confirm the immunostimulatory activity of Oligos 1-8 (Example 8) invivo, a dose of 5 mg/kg of oligonucleotides was injectedintraperitoneally to Balb-C mice. The mice were sacrificed 72 hourspost-administration, spleens were removed, blotted to dryness, andweighed. Change in spleen weight in treated and untreated mice was usedas a parameter for immunostimulatory activity.

Administration 5 mg/kg dose of Oligo 1 caused about 40% increase inspleen weight compared with the control mice that received PBS.Administration of Oligos 2 and 4 also caused about 50% increase inspleen weight. Administration of Oligo 3 caused no difference in spleenweight compared with control mice. These results further support theobservation that Oligo 3, in which 5′-end was blocked, had significantlyless immunostimulatory activity compared to oligos that had accessible5′-end. These results were also confirmed with the administration ofOligos 5-8. Administration of Oligos 5, 6, and 8 caused about 40-50%increase in spleen weight, whereas no change in spleen weight wasobserved following the administration of Oligo 7.

The above results suggest that the immunostimulatory activity ofPS-oligos containing a CpG motif is significantly minimized if the5′-end of the oligo is not accessible. This loss in immunostimulatoryactivity of Oligos 3 and 7 cannot be explained based on nucleasestability, as both oligos have two 3′-ends and are not more susceptibleto 3′-exonuclease degradation than are Oligos 1, 2, 5, and 6, which haveone 3′-end. PS-Oligos 4 and 8, which have their 3′-ends blocked and arevery stable to degradation by exonucleases, showed similarimmunostimulatory activity. Oligos 4 and 8 may show sustainedimmunostimulatory activity due to their increased in vivo stability,which is not evident in the present study as mice were sacrificed atonly 72 hours after administration. Studies are in progress in whichmice will be sacrificed at times later than 72 hours afteradministration.

The results described here are intriguing and suggest that the 5′-end ofCpG-PS-oligos is critical for immunostimulatory activity. As discussedhere, we have shown that substitution of deoxynucleosides in 5′-flankingregions by modified 2′- or 3′-substituted ribonucleosides resulted inincreased immunostimulatory activity. In addition, substitution ofdeoxynucleosides immediately upstream (5′-end) to the CpG motif caused asignificant suppression and substitution of deoxynucleosides immediatelydownstream (3′-end) to the CpG motif had no effect on immunostimulatoryactivity. Taken together, these results suggest that the enzyme/receptorresponsible for the immunestimulation recognizes the CpG motif in oligosfrom the 5′-end and requires accessibility to the 5′-end.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

What is claimed is:
 1. A method for modulating the immunostimulatoryeffect of an immunostimulatory oligonucleotide compound having animmunostimulatory dinucleotide selected from the group consisting ofC*pG, CpG*, and C*pG*, wherein C is 2′-deoxycytidine, C* is selectedfrom 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, and4-thiouracil, G is 2′-deoxyguanosine and G* is selected from7-deazaguanosine and 6-thioguanosine, comprising introducing a1′,2′-dideoxyribose, C3-linker, Spacer 9, Spacer 18,β-L-deoxynucleoside, or 2′-O-propargyl-ribonucleoside into the 5′ and/or3′ side of the immunostimulatory dinucleotide.
 2. The method accordingto claim 1, comprising introducing into the oligonucleotide a1′,2′-dideoxyribose.
 3. The method according to claim 1, comprisingintroducing into the oligonucleotide a C3-linker.
 4. The methodaccording to claim 1, comprising introducing into the oligonucleotide aSpacer
 9. 5. The method according to claim 1, comprising introducinginto the oligonucleotide a Spacer
 18. 6. The method according to claim1, wherein the C3 linker is 2-aminobutyl-1,3-propanediol.
 7. The methodaccording to claim 1, comprising introducing into the oligonucleotide aβ-L-deoxynucleoside.
 8. The method according to claim 1, comprisingintroducing into the oligonucleotide a 2′-O-propargyl-ribonucleoside. 9.The method according to claim 1, wherein G* is 7-deazaguanosine.
 10. Animmunostimulatory oligonucleotide produced by the method according toclaim 1.